Restriction endonucleases are enzymes that occur naturally in certain unicellular microbes—mainly bacteria and archaea—and that function to protect those organisms from infections by viruses and other parasitic DNA elements. These enzymes bind to specific sequences of nucleotides (‘recognition sequence’) in double-stranded DNA molecules (dsDNA) and cleave the DNA, usually within or close to the recognition sequence, disrupting the DNA and triggering its destruction. Restriction endonucleases commonly occur with one or more companion enzymes termed modification DNA methyltransferases. DNA methyltransferases bind to the same sequences in dsDNA as the restriction endonucleases they accompany, but instead of cleaving the DNA, they alter it by the addition of a methyl group to one of the bases within the sequence. This modification (‘methylation’) prevents the restriction endonuclease from binding to that site thereafter, rendering the site resistant to cleavage. Methyltransferases function as cellular antidotes to the restriction endonucleases they accompany, protecting the cell's own DNA from destruction by its restriction endonucleases. Together, a restriction endonuclease and its companion modification methyltransferase(s) form a restriction-modification (R-M) system, an enzymatic partnership that accomplishes for microbes what the immune system accomplishes, in some respects, for multicellular organisms.
A large and varied class of restriction endonucleases has been classified as ‘Type II’ class of restriction endonucleases. These enzymes cleave DNA at defined positions, and when purified can be used to cut DNA molecules into precise fragments for gene cloning and analysis.
New Type II restriction endonucleases can be discovered by a number of methods. The traditional approach to screening for restriction endonucleases, pioneered by Roberts et al. and others in the early to mid 1970's (e.g. Smith, H. O. and Wilcox, K. W., J. Mol. Biol. 51:379-391 (1970); Kelly, T. J. Jr. and Smith, H. O., J. Mol. Biol. 51:393-409, (1970); Middleton, J. H. et al., J. Virol. 10:42-50 (1972); and Roberts, R. J. et al., J. Mol. Biol. 91:121-123, (1975)), was to grow small cultures of individual strains, prepare cell extracts and then test the crude cell extracts for their ability to produce specific fragments on small DNA molecules (see Schildkraut, I. S., “Screening for and Characterizing Restriction Endonucleases”, in Genetic Engineering, Principles and Methods, Vol. 6, pp. 117-140, Plenum Press, NY, N.Y. (1984)). Using this approach, about 12,000 strains have been screened worldwide to yield the current harvest of almost 3,600 restriction endonucleases (Roberts, R. J. et al., Nucl. Acids. Res. 33:D230-D232 (2005)). Roughly, one in four of all strains examined, using a biochemical approach, show the presence of a Type II restriction enzyme.
An in silico screening technique to identify restriction-modification systems has also been described and has been successfully used to identify novel restriction endonucleases (US-2004-0137576-A1). This method relies on identifying new methylases by their consensus sequences. Methylases have much more conservation of amino acid sequence, because they all must bind the methyl donor cofactor S-adenosyl methionine (SAM) and bind the nucleotide to be methylated, either an adenine or a cytosine base, and then perform the methyl transfer chemistry. Although there are several classes of methyltransferases, there are many sequenced examples of methylases and these have well conserved motifs that can be used to identify a protein sequence in a database as a methylase. In this method, identifying restriction endonucleases relies on testing any or all open reading frame (ORF) protein sequences located near the identified methylases.
Since the various Type II restriction enzymes appear to perform similar biological roles and share the biochemistry of causing dsDNA breaks, it might be thought that they would closely resemble one another in amino acid sequence. Experience shows this not to be true, however. Surprisingly, far from sharing significant amino acid similarity with one another, most enzymes appear unique, with their amino acid sequences resembling neither other restriction enzymes nor any other known proteins. Thus the Type II restriction endonucleases seem either to have arisen independently of each other during evolution or to be evolving very rapidly thereby losing apparent sequence similarity, so that today's enzymes represent a heterogeneous collection rather than one or a few distinct families.
Restriction endonucleases are biochemically diverse in their function: some act as homodimers, some as monomers, others as heterodimers. Some bind symmetric sequences, others asymmetric sequences; some bind continuous sequences, others discontinuous sequences; some bind unique sequences, others multiple sequences. Some are accompanied by a single methyltransferase, others by two, and yet others by none at all. When two methyltransferases are present, sometimes they are separate proteins; at other times they are fused. The orders and orientations of restriction and modification genes vary, with all possible organizations occurring. Given this great diversity among restriction endonucleases, it is perhaps not surprising that it has not been possible to form consensus sequences that can be used for in silico searches that are able to identify Type II restriction endonucleases, as has been successfully done for DNA methyltransferases. Thus there is no general common amino acid sequence motif(s) that can be used to identify restriction endonucleases from translated raw DNA sequence ab initio.
Although restriction endonucleases lack conserved sequence motifs and generally have highly diverged DNA and amino acid sequences, some restriction endonucleases are in fact related to one another and, though they may diverge in function, these endonucleases or families of related endonucleases share significant sequence similarity with one another. The key to unlocking these families of endonucleases is to obtain the sequence of one of the members of the related enzymes; from this sequence the other members of the family can be identified. With the advent of whole genome sequencing, many prokaryotic DNA sequences, and from the DNA sequence many amino acid sequences, have become available. Thus there are many amino acid sequences in the database with no known function. This pool of sequences undoubtedly contains numerous restriction endonucleases. The problem is how to identify which genes encode restriction endonucleases, and then how to characterize the function of these genes.